Fecal Elastase-1 in Healthy Children Up To 2 Years of Age: a Cross-sectional Study

Abstract Background Fecal elastase-1 (E-1) levels in infants and young children may be expected to differ from those in adults and older children because of the immaturity of the gastrointestinal tract and the specificity of their diet. Despite the availability of data describing E-1 levels in the stools of preterm infants, older children, adults and subjects with malabsorption, there is still a lack of data regarding E-1 in healthy infants and toddlers. The aim of this cross-sectional study was to evaluate fecal E-1 concentrations in infants and children from 1 up to 24 months of age. Material and methods E-1 was measured in 160 healthy subjects aged 1-24 months (8 groups of 20: aged 1-3, 4-6 months, etc.) using an enzyme-linked immunosorbent assay (ELISA). Results Fecal E-1 concentrations ranged from 200 to 1695 μg/g of feces. No child had a fecal E-1 level below 200 μg/g of feces. Fecal E-1 concentrations did not significantly differ between age groups. However, fecal E-1 levels in the first 3 months were lower than in the second year of life (1-3 months vs 13-24 months, p=0.0230). A statistically significant correlation between the E-1 concentration and age was found (p=0.0007, r=0.2639; however, it does not affect the cut-off level of the reference values). The trend was rather exponential. Fecal E-1 values reached a plateau around the age of 6-10 months. Conclusions Our study has shown that the fecal E-1 test can be reliably applied in infants and toddlers to confirm normal exocrine pancreatic function. However, within the first months of life fecal E-1 concentrations may be lower than later in life.

Słowa kluczowe: badanie kału, funkcja zewnątrzwydzielnicza trzustki, test funkcji trzustki, zdrowe niemowlęta INTRODUCTION Elastase-1 (E-1) is a marker of exocrine pancreatic insu ciency that is widely used owing to its high speci city and sensitivity [1][2][3][4][5]. Low fecal E-1 concentrations occur in diseases involving pancreatic dysfunction, such as: cystic brosis, diabetes, chronic pancreatitis, pancreatic cancer, coeliac disease and human immunode ciency virus infection [6][7][8][9][10]. e abundance of E-1 in stools depends on a person's diet and age. Our previous research revealed that abstaining from meat results in a signi cant decrease in E-1 [11,12] and that E-1 levels in the elderly are lower compared with young people, re ecting the natural aging of the pancreas [13]. Many di erent changes may be expected to be found in infants and young children since their gastrointestinal tract is immature and their diet di ers from that of adults. However, the available data regarding fecal E-1 concentrations in healthy children, and especially in infants, is scarce. In fact, most of the studies conducted to-date described the evolution observed in the secretion of pancreatic enzymes over the rst days a er birth in preterm infants and measuring E-1 levels in their meconium [14][15][16]. Low E-1 concentrations in the meconium and their increase in feces in the rst weeks of life was noted in these studies. However, research which would involve representative groups of healthy infants and toddlers is still lacking.
AIM e aim of this cross-sectional study was to evaluate fecal E-1 concentrations in infants and children from 1 up to 24 months of age.
All the children remained under the care of the investigator (MFW) for at least three years a er E-1 was measured. No signs and symptoms of neither exocrine pancreatic insu ciency or other gastrointestinal diseases potentially in uencing pancreatic secretion have been observed in any of the study participants. e protocol of the investigation was approved by the Ethical Committee of Poznań University of Medical Sciences, Poznań, Poland (decision no. 1275/05). Written informed consent was obtained from all the children's parents. e study was carried out in accordance with the revised Declaration of Helsinki.

Methods
In every child the Z-score for body weight was calculated [17]. Fecal E-1 concentrations were measured in stool samples with a commercially available monoclonal antibody kit (ELISA; ScheBo BioTech, Giessen, Germany) [6,18]. Results were expressed as μg/g of feces.

StaƟsƟcal methods
All the statistical analyses were performed in the Statistica 12.0 so ware environment (StatSo , Inc., Tulsa, USA) and Stata/IC 15.0 64 bit for Windows (StataCorp LP, Lakeway Drive, USA). Normality was determined using the Shapiro-Wilk test. e normal distribution mean and standard deviation (SD) are reported for variables. Medians and 1st-3rd quartiles are given for non-normal variables. Di erences between multiple groups were assessed using the Kruskal-Wallis test and one-way analysis of variance with post-hoc testing (Bonferroni-corrected). e linear correlation between E-1 concentrations and age were analyzed using Pearson's test.
E-1 results were smoothed for graphic presentation using the LOWESS method (Stata/SE 15.0 64 bit for Windows, Tulsa, USA).
Values of p<0.05 were considered to be statistically signi cant.

RESULTS
e anthropometric parameters describing the subjects are presented in Table 1. No statistically signi cant di erences between Z-scores for body weight and sex were found between the groups (Table I).
Fecal E-1 concentrations ranged from 200 to 1695 μg/g of feces. No child had fecal E-1 levels below the cut-o level (200 μg/g) suggestive of abnormal exocrine pancreatic function.
Fecal E-1 concentrations did not signi cantly di er between age groups (Table II). However, fecal E-1 levels in the rst 3 months were lower than in the second year of life (median [1 st -3 rd quartile], 790 μg/g [527-920] vs 900 μg/g [728-1070]; p=0.0230). A statistically signi cant correlation between E-1 concentration and age was found (p=0.0007, r=0.2639; however, it does not a ect the cuto level of the reference values). e trend observed was rather exponential (Figure 1). Fecal E-1 values reached a plateau around the age of 6-10 months.
DISCUSSION e topic of our study was the evaluation of fecal E-1 concentrations in healthy children aged 1-24 months.
is has been the rst time when in such a large cohort of healthy infants and young children (n=160) exocrine pancreatic function was studied using reliable indirect pancreatic function tests. It should be emphasized that this is the rst study to create reference values of E-1 in healthy infants and toddlers.
E-1 concentrations in all children were within the normal range for adults. Similarly, Nissler et al. reported that 96.8% of the infants they studied had higher fecal E-1 levels a er the second week of life than the adult lower limit of normal values, independent of the gestational age at which they were born [16]. However, their study comprised predominantly patients younger than 3 months with only a few older infants. It should be noted that the youngest child in the present study was older than 4 weeks and no meconium assessments were made. We did not include neonates, because extensive data are already available for this age group [14][15][16][19][20][21]. Kori et al. noted that in all the samples analyzed (n=63) regardless of gestational age, the E-1 level was below normal range (200 μg/g of feces; mean (SD) 45.9 μg/g of feces (51.1)) and found that E-1 normalization followed day 3 in term newborns [14]. Also in preterm babies studies described a low E-1 concentration in their rst few days of life (median (range): 89 μg/g of feces (3-539) at day 2) which gradually increased on subsequent days (median (range): 164 μg/g of feces (3-600 at day 5) [15].
In our study fecal E-1 levels were lower within the first months of life. Later they reached a plateau corresponding to normal adult levels. Also Terbrack et al. described a slight tendency of E-1 levels to increase with age and similarly high enzyme concentrations in a population of older infants and children (the mean was 763 μg/g) [19]. However, a detailed analysis was performed only for the first 4 months of life (4 groups aged 1, 2, 3 and 4 months). Older subjects were divided into 2 groups with a large age span (the first group − from 5 months to 1 year, the second − from 1 year to 14 years). e increase of values observed within the rst months of life in the present study may be due to the development of the pancreas. Although the pancreas begins to produce enzymes  Ns.
The number of subjects in each group was 20 W każdej grupie wiekowej było 20 dzieci already in the 20 th week of gestation, newborns secrete fewer pancreatic enzymes than older children (for example, amylase levels are undetectable, while lipase is produced on a level of less than 10% of childhood levels) [22,23]. It should be noted that the ontogeny of E-1 in infants is still unknown. e documented di erences in fecal E-1 concentration between age subgroups might be related to weaning and expanding infants' diet (especially by the 6 th month of life). In the data available, the normal levels of fecal E-1 were found sooner in infants who started enteral feeding earlier [14,15]. It should, however, be noted that infants are fed a very speci c (high fat) diet based exclusively on milk fat. On the other hand extending their diet may stimulate enzyme production and secretion, which is supported by studies on adults that have shown that an abstinence from meat may lead to a decrease in fecal E-1 concentration [11].
It should be stressed that the abundant data concerning fecal E-1 concentrations in children with cystic brosis [24][25][26][27] or preterm infants [14,15,21] cannot be extrapolated from healthy infants. All the studies conducted on healthy children so far involved heterogenous age groups (from birth to almost adulthood) with low numbers of infants and toddlers [28,29]; this renders the results virtually uninterpretable beyond the neonatal and early post-neonatal period.
Although the present study comprises a large and representative group of infants and young children aged up to 24 months, it also has some limitations which includes the cross-sectional character of the study with the lack of longitudinal follow-up of fecal E-1 concentrations, the absence of information on the weaning status, and the lack of correlation of fecal E-1 levels to energy and nutrient intake.

CONCLUSIONS
Our study has shown that the fecal E-1 test can be reliably applied in infants and toddlers to con rm normal exocrine pancreatic function. However, within the rst months of life fecal E-1 concentrations may be lower than later in life.